| 1. |
- Lundberg, Peter, et al.
(author)
-
Nuclear magnetic resonance studies of cellular metabolism
- 1990
-
In: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 191:2, s. 193-222
-
Journal article (peer-reviewed)abstract
- Nuclear magnetic resonance (NMR) spectroscopy was described in 1946 (1,2), initially as a method that had appeal only for nuclear physicists who used it to accurately determine nuclear magnetic moments. Thissituation changed rapidly, however, when it was demonstrated that the NMR frequency for the same nucleus in different chemical compounds was different (3). For example, two separate signals are observed in a 14N NMR spectrum of a solution of NH,NO,, representing the NH: and NO; ions, respectively (4). Since individual atoms within one molecule also give rise to resolved signals (5) it became clear that the NMR technique held great analytical potential, in particular since the spectra can be recorded in such a way that the area under a signal is directly proportional to its concentration. Such phenomena and various theoretical aspects of NMR are currently quite well understood (6,7). Because of these features NMR has become the foremost spectroscopic method for the analysis of all sorts of chemical compounds.
|
|
| 2. |
- Toomik, Reet, et al.
(author)
-
A potential pitfall in protein kinase assay: phosphocellulose paper as un unreliable adsorbent of produced phosphopeptides
- 1992
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 204:2, s. 311-314
-
Journal article (peer-reviewed)abstract
- The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.
|
|
| 3. |
- Bondeson, K, et al.
(author)
-
Lactose repressor-operator DNA interactions : kinetic analysis by a surface plasmon resonance biosensor.
- 1993
-
In: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 214:1, s. 245-51
-
Journal article (peer-reviewed)abstract
- Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Lennerstrand, Johan, et al.
(author)
-
A Method for Combined Immunoaffinity Purification and Assay of HIV-1 Reverse Transcriptase Activity Useful for Crude Samples
- 1996
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 235:2, s. 141-152
-
Journal article (peer-reviewed)abstract
- Detection of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity in crude specimens was greatly enhanced using a novel capture RT assay. Eighteen different monoclonal antibodies (Mabs) raised against purified HIV-1 RT were tested for their ability to bind to HIV-1 RT without affecting its activity. The anti-HIV-1 RT Mabs were immobilized on plastic macrobeads and used as solid carriers in the capture RT assay. The assay system first involved RT's adherence to the immobilized Mabs. Nonspecific enzymes and other impurities were removed by a simple wash after which the RT reaction mixture was added. Substrate and product were finally separated by a wash of the beads. Practically all radioactivity incorporated into DNA (>98%) was recovered on the bead. The Michaelis-Menten constants and the saturation velocity values for the nucleotide substrate were similar for free and immobilized RT. The reaction mechanism for the immobilized RT is discussed. When comparing the function of this assay with more conventional soluble RT assays for samples consisting of recombinant HIV-1 RT mixed with an extract of peripheral blood lymphocytes (PBL), an almost 100-fold higher sensitivity was found. The capture RT assay had the capacity to recover approximately 80% of the RT activity added to an extract of 1 x 10(7) PBL cells/ ml. A strong correlation (r = 0.947) between the results obtained with this assay and a HIV-1 p24 enzyme-linked immunosorbent assay was found, when samples from a collection of 16 HIV strains propagated in cell culture were analyzed.
|
|
| 8. |
- Markgren, Per-Olof, et al.
(author)
-
Screening of compounds interacting with HIV-1 proteinase using optical biosensor technology
- 1998
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 265:2, s. 340-350
-
Journal article (peer-reviewed)abstract
- A high-resolution optical biosensor assay for screening of low-molecular-weight compounds, using an immobilized protein target, has been developed. HIV-1 proteinase was immobilized on the sensor surface by direct amine coupling and a variety of inhibitors and noninteracting reference drugs were applied to the sensor surface in a continuous how of buffer. The procedure did not require intrinsic reporter groups, substrates, inhibitors, or other ligands for detection. By using a reference protein, the signal could be corrected for the relatively large background signal caused by differences in dimethyl sulfoxide concentration between running and sample buffers. Substances binding with high affinity (K-i in nM range) required efficient regeneration of the sensor surface and washing of the injection system between sample cycles to get consistent results. Analysis was simplified by using report points, extracted during both association and dissociation phases, and a simple graphical display of data. The optimized assay could correctly distinguish HIV-1 inhibitors from other compounds in a randomized series, indicate differences in their interaction kinetics, and reveal artifacts due to nonspecific signals, incomplete regeneration, or carryover. The method is expected to be generally applicable to secondary screening of low-molecular-weight compound libraries with proteins as targets.
|
|
| 9. |
- Nilsson, Peter, et al.
(author)
-
Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers
- 1999
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 269:1, s. 155-161
-
Journal article (peer-reviewed)abstract
- The effect on oligonucleotide–template duplex stability upon cohybridization of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexamer modules and sensor chip-immobilized template DNA strands were designed for analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the determination of apparent duplex affinities from equilibrium responses. The results showed that the hybridizational stability of modular hexamer pairs is strongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in apparent affinities could be observed for adjacent modular oligonucleotide pairs compared to affinities determined for single hexamer oligonucleotide hybridizations. Interestingly, also for coinjections of different module combinations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be useful for the selection of appropriate modules and working concentrations for use in different modular hybridization applications.
|
|
| 10. |
- NILSSON, PETER, et al.
(author)
-
REAL-TIME MONITORING OF DNA MANIPULATIONS USING BIOSENSOR TECHNOLOGY
- 1995
-
In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 224:1, s. 400-408
-
Journal article (peer-reviewed)abstract
- The potential of real-time biospecific interaction analysis technology for applications in molecular biology is described. DNA fragments are immobilized onto a biosensor surface using the high-affinity streptavidin-biotin system and subsequently used to monitor different unit operations in molecular biology, e.g., DNA strand separation, DNA hybridization kinetics, and enzymatic modifications. A model system comprising six oligonucleotides was used, which can be assembled into a 69-bp double-stranded DNA fragment. Using this system, the biosensor approach was employed to analyze multistep solid-phase gene assembly and the performance of different enzymes routinely used for the synthesis and manipulation of DNA. In addition, a concept for the determination of single-point mutations in DNA samples is described.
|
|
